19
20
21
22
2
5
6
3
4
1 Title: Comparison of the pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) patterns for subtyping Salmonella
Choleraesuis isolates from swine field in southern Taiwan
7 TSAI-HSIN CHIU
1
, JEN-CHIEH PANG
1
, WEN-ZHE HWANG
1
, MING-HUEI LIAO
2
,
8
9
GAN-NAN CHANG
2
, AND HAU-YANG TSEN
1*
.
10
11
1
Department of Food Science, National Chung-Hsing University, Taichung, Taiwan, R.O.C.
12 2 epartment of Veterinary Medicine, National Ping-tung University, Ping-tung, Taiwan, R.O.C.
13
14
15
*
Corresponding author: Hau-Yang Tsen, Professor
16
17
Department of Food Science,
National Chung-Hsing University,
18 Taichung, Taiwan, R.O.C.

1 Abstract
2 Salmonella Choleraesuis is one of the major bacterial pathogens often isolated from
3 diarrheagenic swine in Taiwan. In the study, pulsed field gel electrophoresis (PFGE)
4 and randomly amplified polymorphic DNA analysis (RAPD) were used for the
5 subtyping of ninety-five S. Choleraesuis strains isolated from swine with diarrhea and
6 systemic infection during 2000~2002. The swine were grown in the swine fields of
7 southern Taiwan. For PFGE, XbaI, NotI and SpeI restriction enzymes were used for
8 chromosomal DNA digestion. The results showed that, for these 95 Salmonella strains,
9 63 PFGE patterns combinations were found. Of them, pattern X1N1S1, was the major
10 subtype since 25 strains belong to this pattern. Strains of this pattern may be the most
11 epidemic strains. On the other hand, RAPD analysis generated 74 patterns in 95 isolated
12 strains. Comparison of the PFGE and RAPD patterns, RAPD could subdivide strains
13 within a PFGE type. Thus, RAPD was the most discriminative tool for subtyping of
14 Salmonella Choleraesuis, if suitable primer was selected for typing.
15
16
17 Key words: Salmonella Choleraesuis, PFGE, RAPD.
18
19

1 INTRODUCTION
2 Salmonella Choleraesuis is a host-adapted, facultative, intracellular pathogen
3 bacterium (Wilcock et al., 1992; Jeffrey et al., 1996). It is a common pathogen for both
4 human and animals, such as porcine. The salmonellosis caused by S . Choleraesuis has a
5 high predilection for causing systemic infection in humans (Cohen et al., 1987), the
6 fatality rate 2-3 times of typhoid. Infection by S.
Choleraesuis was uncommon in
7 humans, but common and important for swine. Since S. Choleraesuis has been recently
8 the most important Salmonella serovar, which cause severs diarrhea cases for porcine in
9 Taiwan. The incidence of serovar Choleraesuis infection is rather high. Among the
10 Salmonellae in Taiwan, the frequency of serovar Choleraesuis infection is only second
11 to the serovar Typhimurium and serovar Schwarzenground (Chiu et al., 1999).
12 Many methods have been used for the molecular typing of Salmonella spp., such as
13 arbitrarily primed PCR (AP-PCR)(Fadl, et al., 1995), ribotyping (Thong et al. 1995;
14 Lagatolla et al., 1996; Martina et al., 1996; Toshio et al., 2002), IS 200 profile (Martina
15 et al., 1996; Yevs et al., 1995), enterobacterial repetitive intergenic sequence PCR
16 (ERIC-PCR) (Swanenburg, 1998; Toshio, 2002; Yves, 1995), infrequent-restriction-site
17 PCR (IRS-PCR) (Chiu, 2002), pulsed-field gel electrophoresis(PFGE) (Martina, 1996;
18 Toshio, 2002; Tsen, 2002) and randomly amplified polymorphic (RAPD) (Niwat et al.,
19 2000). Those methods also have been commonly used for the differentiation and typing

1 of other bacterial strains. In general, any subtyping method must have high
2 discriminative power for analysis (Arbeit, 1995). Subtyping or strain classification was
3 generally accomplished by use of a number of different approaches, and all of those
4 methods should meet several criteria in order to be broadly useful (Oliver, 1999). For
5 some of the above described, such as IRS-PCR, it did not differ significantly in the
6 distribution of their patterns from different species, in different years, and difference
7 antibiogram isolates (Chiu, 2002);. Martina et al. (1996) report that IS 200 profile should
8 not be included in a basis set of molecular methods for typing S. choleraesuis isolates,
9 even if IS 200 elements occasionally occur in S. choleraesuis ; Ribotyping method, the
10 discriminatory power of the method depends on the choice of the gene probe, and it was
11 not suitable for different S.
Choleraesuis isolates.
12 In this study, we evaluated the common molecular typing methods and elucidated the
13 clonal relationship and genetic diversity for the Salmonella Choleraesuis strains isolated
14 from diarrheagenic swine in the swine fields of southern Taiwan. This study may
15 possible enable us to find the prevalent subtypes or most epidemic strains which caused
16 swine diarrhea and infection.
17
18 MATERIAL AND METHODS
19 Bacterial strains.

1 A total of 95 strains of S.
Choleraesuis obtained from diarrhea or infective swine in
2 the swine fields of southern Taiwan. There were isolated from 2000 to 2002. Those S.
3 Choleraesuis strains were obtained from the Department of Veterinary Medicine,
4 National Ping-Tung University, Ping-Tung, Taiwan. S.
Chloeraesuis ATCC 13312 was
5 used as reference strain. All the trains collected were confirmed by biochemical tests
6 using API20E commercial kit, and serotyping by the Kauffman-White scheme. All the
7 strains were grown on Tryptic Soy broth (TSB) and were stored frozen at -80℃ for
8 future transfer.
9 Genome typing by PFGE
10 Methods described by Tsen et al. (2000) were modified for PFGE typing. Generally,
11 S . Choleraesuis isolates were grown in 5 ml of TSB broth at 37℃ for 4 h. After
12 harvesting, washing and resuspeding, these cells were heated to 50℃, mixed with 0.3
13 ml of 2% low melting point agarose in lysozyme buffer (10mM Tris-HCl, pH 7.2;
14 50mM NaCl, 100mM EDTA; 0.2% sodium deoxycholate and 0.5% N-lauryl-sarcosine).
15 The mixture was poured into the slots of a plastic mould (CHEF DRII, Biorad, Hercules,
16 California, USA) and the agarose plugs obtained were transferred into 3 ml of lysozyme
17 solution containing 2 mg ml
-1
and subjected to cell lysis(37 ℃ , 24h). The lysis buffer
18 was then replaced by 3 ml of proteolysis solution (0.1M EDTA, 1% lauroylsarcosine,
19 pH 8.0, 0.2% sodium deoxylate, 0.1 mg ml
-1
proteinase K) and proteolysis was carried

1 out at 50℃ overnight. Finally, the agarose plugs were washed with wash buffer (20mM
2 Tris-HCl; 50mM EDTA, pH 8.0) at 4℃. Restriction digestion was performed by
3 placing a 2-mm slice of each plug into 100
 l of restriction buffer (100mM NaCl,
4 50mM Tris-HCl, 10mM MgCl
2
, 1mM dithiothreitol). After 15 min, the plug was put
5 into 100
 l of the same buffer containing 1 mg ml
-1
BSA and 20 units of Xbal
Ⅰ, or 10
6 units of Not
Ⅰand
Spe I.These restriction enzymes were purchased from Bio-Labs
7 (Beverly, Massachusetts, USA). After 3 h incubation at 37℃, the plugs were then
8 placed into the slots of 1.2
％ agarose gel. Electrophoresis was performed by the use of
9
CHEF DRⅡ PFGE system (Bio-Rad). The conditions used were 200V for 28 h at 12℃
10 with pulse time ranging from 2 to 48 s for Xba
Ⅰdigested DNA, 200 V for 26 h at 12℃
11 with pulse time ranging from 1 to 12 s for Not
Ⅰdigested DNA and 200 V at 12℃ with
12 two steps pulse time ranging from 7-12 s, 13 hr and 20-40 s, 11 hr for Spe I digested.
13
Bacteriophage λ DNA concatiamers (Bio-Labs) were used as molecular weight markers.
14 The gel were stained with ethidium bromide and photographed.
15 The PFGE patterns obtained were analyzed by visual assessment and the dendrogram
16 was generated from NTSYS 2.0. The similarity of fragments between two isolates was
17 scored by Dice coefficients using formula F= 2 nxy /( nx + ny ), where F is the coefficient of
18 similarity. An F of 1.0 indicates that two isolates have identical patterns.
19 RAPD-polymerase chain reaction conditions

1 Chromosomal DNA of the 95 strains of S . Choleraesuis was extracted. The guidelines
2 from commercial kit (Viogen, Taiwan) were followed. All the DNA samples were
3 amplified using a selected primer, ie, (5’-d(GTTTCGCTCC)-3’), for RAPD typing.
4 This primer has been previously described and used by Niwat et al.(1996).
5 Amplification was performed in a 25
 l reaction mixture contained 10
 l of 5

M RAPD
6 primer-2, 100 ng of template DNA, l00 mM dNTP, 1.5 mM MgCl
2
, 1 × reaction buffer
7 and 0.5U Supertherm polymerase(Bertec, Taiwan). The mixtures were subjected to PCR.
8
DNA was amplified 40 cycles of 20s at 94℃; 20s at 36℃ and 30s at 72℃. 10  l of the
9 PCR product was assayed by electrophoresis with 2 ％ gel and photographed
10 Calculation of discriminatory indices.
11 The discriminatory value for PFGE and RAPD typing methods were calculated as an
12 index of discrimination ( D ) according to Hunter and Gaston (1988). It served for the
13 comparison of the different methods and for the selection of the most discriminatory
14 system for the molecular differentiation of S.
Choleraesuis isolates.
15
16 RESULTS
17 Macrorestriction analysis .
18 When chromosomal DNAs from all the 95 S.
Choleraersuis strains were digested
19 with Xba
Ⅰ, a total of 29 PFGE patterns were identified (Table 1). The patterns consist of

1 11 to 15 fragments with molecular sizes ranging between 48.5 and 533.4 kb (Fig 1). The
2 coefficients of similarity ( F ) for these 95 Salmonella isolated are ranged from 0.400 to
3 1.000 indicating that considerable genetic diversity is present in these strains (Fig 2A).
4 52 in 95 strains (54.7%) belong to a pattern X1, which is the major pattern. All the S .
5 Choleraesuis strains from systemic infection belong to this pattern. Not I digestion yield
6 25 different patterns (Table 1). These patterns consist of 30 to 35 fragments with
7 molecular sizes ranging 48.5 and 242.5 kb. This enzyme produced a large number of
8 smaller fragments. F value of the NotI digested patterns ranged from 0.733 to 1.000,
9 and 62 in 95 strains (65.3%) belong to a pattern N1 which is the major pattern. The
10 discriminatory index of Not I was lower. When Xba I and Not I patterns were combined
11 for analysis, 46 PFGE patterns were found. The patterns combination of X1N1, which
12 encompass 41 strains, ie, 43.2% of the 95 strains, was the major subtype. Strains in the
13 subtype, ie, X1N1, were genetically highly similar or clonally highly related. Almost all
14 the isolated strains from systemic infection belonged to this type, X1N1.
15 RAPD.
16 Although RAPD may be unreliable due to its poor reproducibility, it has been
17 successfully used for the subtyping of S . Choleraersuis. A total of 95 reproducible
18 amplification products were sufficiently polymorphic to allow the differentiation of the
19 strains under study. Depending on the selected primer, 74 patterns were obtained (Table

1 1). The patterns fragments were between 300 and 2000 bp (Fig 3). And those strains
2 used for RAPD typing were shown to have four major bands. When the RAPD patterns
3 and PFGE patterns were combined for subtyping of those isolates, it was found that
4 strains in X1N1 type could be further discriminated.
5 Comparison of PFGE and RAPD typing methods.
6 D value was a single numerical index for discrimination power. D value could be
7 used in comparison of the typing methods. The D value of PFGE patterns which
8 combine with Xba I and Not I digestion was 0.6974, and the RAPD pattern was
9 calculated to be 0.9935. Based on the results, RAPD obtained a highly D value
10 confirmed the usefulness for the epidemiology analysis of S . Choleraesuis. When
11 combination those two typing methods, the discriminatory index was calculated to be
12 0.999.
13
14 DISCUSSION
15 Swine infection due to S.
Choleraesuis was has been common in Taiwan. Based on
16 the face that this strain could transmit between humans and swine, it was urgent us to
17 perform the epidemiologic study for of S.
Choleraesuis. Discriminatory power,
18 rapidness, simplicity, and reproducibility are important attributes in any typing method.
19 In this study, we used PFGE and RAPD for molecular typing of S, Choleraesuis.

1 When restriction digestion patterns were analyzed by PFGE, epidemiologically
2 related isolates usually generated indistinguishable or, on occasion, closely related
3 fragment patterns. For most of the common bacterial pathogens, the validity of PFGE
4 for molecular typing is well established (Arbeit, 1995). The discriminatory value of
5 microrestriction analysis could be increased as several suitable restriction enzymes were
6 used (Garaizar, 2000). In this study, Xbal I could yield higher discriminatory than Not I,
7 but both enzymes could not differentiate within the species level very well. However,
8 the results may indicate that prevalent subtypes of S. Choleraesuis from swine isolates
9 do exist. Because of PFGE typing was reproducible and discriminatory, if suitable
10 enzymes were found, this method may be a powerful tool for the epidemiological
11 typing of S.
Choleraesuis isolates.
12 RAPD typing may be limited by poor reproducibility for molecular typing, it has
13 been successfully used for subdivide Salmonella erterica subsp. erterica both inter- and
14 intra-serovars. (Niwat, 2000). In this study, we have obtained the highest discriminatory
15 value and reproducibility for RAPD analysis. As this method was used for subdivide of
16 the PFGE patterns, the major PFGE pattern X1N1, could be further differentiated into
17 different patterns. However, for good discriminatory power, it was suggested that
18 suitable primer should be used. This is important for screening and studying of the
19 molecular genetic epidemiology of S.
Choleraesuis isolates. In conclusion, when PFGE

1 and RAPD molecular typing methods were combined, it may give the highest
2 reproducibility and discriminatory power for investigation of Salmonella spp..
3
4 ACKNOWLEDGMENTS
5 This project is supported by the Nation Science Council, Taipei, Taiwan. The project
6 No. is NSC 91-2313-B-005-074.
7
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11
12
13
14
7
8
9
10
15
16
17
18
19
1 profiling, ribotyping, and detection of IS200 for tracing avian isolates of Salmonella
2 typhimurium and S. enteritidis . J. Clin. Microbiol. 33:173-179.
3
4
5
6

1
2
3
X7N7
X8N1
X9N1
X9N11
X9N12
X9N22
X10N9
X10N1
X10N15
X11N10
X12N1
X13N13
X14N1
X15N12
X16N14
X17N1
X1N3
X1N5
X1N6
X1N9
X1N12
X1N16
X1N19
X1N21
X1N23
X2N2
X3N4
X3N8
X3N17
X4N1
X5N1
X6N7
X18N1
X19N1
X20N12
X21N18
X22N1
X23N1
X24N1
X25N1
X26N1
X27N1
X28N1
X28N20
X29N22
Total
Table 1 . PFGE subtyping for the 95 S.
Chloraersuis strains
PFGE Types
X1N1
Strain
SCV30
SCV33, SCV65
SCV35
SCV36
SCV38
SCV47
SCV50, SC
SCV52
SCV53
SCV54
SCV55
SCV59
SCV60
SCV61
SCV62
SCV69
SCV64
SCV70
SCV7, SCV9, SCV10, SCV15, SCV23, SCV29,
SCV31, SCV32, SCV34, SCV37, SCV40, SCV42,
SCV45, SCV46, SCV58, SCV63, SCV67, SCV68,
SCV72, SCV1a~SCV12a, SCV14a~SCV17a,
SCV20a, SCV21a, SCV23a~SCV27a, E55
SCV3
SCV5
SCV8
SCV14, SCV17, SCV20, SCV24
SCV27
SCV41
SCV57
SCV66
SCV19a
SCV2
SCV4
SCV13, SCV18
SCV44
SCV6
SCV11, SCV71
SCV12
SCV16
SCV19
SCV22, SCV13a
SCV26
SCV22a
SCV18a
SCV24
SCV49
SCV39, SCV43
SCV25
SCV28
.
NO. strains
41
2
1
1
1
2
1
1
1
1
1
1
1
1
1
2
1
1
1
2
1
1
1
1
2
1
1
1
1
1
1
1
4
1
1
1
1
1
95
1
1
1
1
1
2
1
1

28
29
30
31
24
25
26
27
32
33
34
20
21
22
23
16
17
18
19
35
36
37
38
9
10
11
12
13
14
15
5
6
7
8
1
2
3
4
B
A
Dice similarity coefficient (%)
Dice similarity coefficient (%)
Fig 2 . Similarity dendrogram obtained from cluster analysis of PFGE macrorestriction patterns of the 95 S . Chloeraersuis isolates. A : Cluster of Xba lI digestion DNA patterns.
B : Cluster analysis on the basis of combined Xba I and Not I restriction enzymes digestion patterns.

13
14
15
16
9
10
11
12
5
6
7
8
1
2
3
4
21
22
23
24
25
17
18
19
20
30
31
32
33
26
27
28
29
34
35
36
37
38
M 1 2 3 4 5 6 7 8 9 10 11 12 13 M
Fig 1.
Xbal I digested DNA patterns for S.
Choleraesuis isolated strains. Lane M: marker
λ ladder. Lane 1-13 represent the PFGE types obtained from S . Choleraesuis strains of
SCV39, 40, 38, 43, 44, 45, 46, 47, 49, 50, 52, 53, 65. Lane 2 was the major pattern, X1.

13
14
15
16
9
10
11
12
5
6
7
8
1
2
3
4
21
22
23
24
17
18
19
20
25
26
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Fig 3.
RAPD profiles of S. Choleraesuis isolates using primer2. Lane M: 100 bp marker ladder; lane 1-14: SCV2, 3, 22, 41, 54, 55, 57, 60, 61, 69, 70, 1a, 16a, 25a
27